WO2020050294A1 - Homologous recombination efficiency increasing agent, and use thereof - Google Patents

Homologous recombination efficiency increasing agent, and use thereof Download PDF

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WO2020050294A1
WO2020050294A1 PCT/JP2019/034705 JP2019034705W WO2020050294A1 WO 2020050294 A1 WO2020050294 A1 WO 2020050294A1 JP 2019034705 W JP2019034705 W JP 2019034705W WO 2020050294 A1 WO2020050294 A1 WO 2020050294A1
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homologous recombination
cells
cell
recombination efficiency
dna
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岡野　栄之
誠司 塩澤
祥 吉松
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学校法人慶應義塾
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
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    • C12N5/06—Animal cells or tissues; Human cells or tissues

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  • the present invention relates to an agent for increasing the efficiency of homologous recombination and its use. More specifically, the present invention relates to an agent for increasing the efficiency of homologous recombination, a method for producing a cell having an increased efficiency of homologous recombination, and a method for producing a cell having genomic DNA modified by homologous recombination.
  • This application claims priority based on Japanese Patent Application No. 2018-165813 for which it applied to Japan on September 5, 2018, and uses those content here.
  • Homologous recombination means that when one double-stranded DNA of two different double-stranded DNAs is cleaved and a homologous DNA sequence exists near the cleavage site, crossover occurs in the homologous sequence, Refers to the exchange of DNA sequences between double-stranded DNA.
  • Genetic modification technology that uses a site-specific nuclease to cause DNA double-strand breaks in any genomic region and uses the DNA repair mechanism is called genome editing, and gene knock-in / knock-out at the cell or individual level is possible. .
  • Non-Patent Document 1 shows an example in which five genes are simultaneously mutated in mouse ES cells using genome editing technology.
  • NHEJ non-homologous end joining
  • HDR homology-directed DNA repair
  • the knock-in technique has an advantage over the knock-out technique in the production of human disease iPS cells and disease model mice.
  • Knock-in technology is also indispensable for advanced genetic modification such as spatio-temporal conditional knockout.
  • ⁇ DNA repair via NHEJ cannot control the DNA sequence to be inserted or deleted.
  • HDR-mediated DNA repair In order to insert a desired DNA fragment into a target site, it is necessary to use HDR-mediated DNA repair.
  • an object of the present invention is to provide a technique for improving the efficiency of homologous recombination.
  • the present invention includes the following aspects. [1] containing, as an active ingredient, a DNA cleavage repair-related protein or a nucleic acid encoding the protein, which has a higher expression level in marmoset cells than a cell of a species having a lower homologous recombination efficiency than a marmoset cell; Agent for increasing homologous recombination efficiency. [2] The species whose homologous recombination efficiency is lower than that of marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. The agent for increasing the efficiency of homologous recombination according to claim 1, which is used.
  • [3] The agent for increasing homologous recombination efficiency according to [1] or [2], wherein the DNA breakage repair-related protein is a RAD51 paralog and a homologous recombination factor involved in susceptibility to breast cancer.
  • [4] The agent for increasing homologous recombination efficiency according to [3], wherein the DNA breakage repair-related protein is selected from the group consisting of BRCA1, BRCA2, RAD51C, and RAD51D.
  • a production method comprising introducing the homologous recombination efficiency increasing agent according to any one of the above.
  • the species whose homologous recombination efficiency is lower than that of marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. Production method according to [5].
  • the species whose homologous recombination efficiency is lower than that of marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat, and chicken.
  • a method for producing a cell in which genomic DNA has been modified by homologous recombination, wherein the cell is a species of a cell having a lower homologous recombination efficiency than a marmoset cell and [4] A production method, comprising: introducing the homologous recombination efficiency increasing agent according to any one of [4], introducing a donor DNA into the cell, and cutting the genomic DNA of the cell. .
  • the species having a lower homologous recombination efficiency as compared to marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat, and chicken. Production method according to [9].
  • a technique for improving the efficiency of homologous recombination can be provided.
  • FIG. 8 is a diagram illustrating an experimental system in Experimental Example 2 for causing homologous recombination at the HPRT locus and calculating homologous recombination efficiency.
  • FIG. 9 shows the results of measuring the expression of the RAD51C gene in marmoset ES cells or human iPS / ES cells by real-time PCR in Experimental Example 2.
  • FIG. It is the result of having measured the expression of the RAD51D gene in the marmoset ES cell or the human iPS / ES cell in Experimental Example 2 by real-time PCR.
  • 5 shows the results of measuring the expression of the RAD51 gene in marmoset ES cells or human iPS / ES cells in real time PCR in Experimental Example 2.
  • FIG. 5 shows the results of real-time PCR measuring the expression of the BRCA1 gene in marmoset ES cells or human iPS / ES cells in Experimental Example 2.
  • 5 shows the results of measuring the expression of the BRCA2 gene in marmoset ES cells or human iPS / ES cells in real time PCR in Experimental Example 2.
  • FIG. 9 shows the results of calculating the homologous recombination efficiency of the HPRT gene when each DNA breakage repair-related protein was expressed in human iPS cells in Experimental Example 3.
  • FIG. 10 shows the results of calculating the homologous recombination efficiency in the HPRT gene in Experimental Example 3 when human iPS cells were expressed in combination with DNA breakage repair-related proteins (BRCA1, BRCA2, RAD51C, and RAD51D).
  • FIG. 9 is a diagram illustrating an experimental system for performing homologous recombination at the Sox2 locus in Experimental Example 4.
  • 9 is a microscope image of cells expressing SOX2 :: Venus fusion protein in the nucleus in Experimental Example 4.
  • 9 shows the results of calculating the Venus-positive colony rate in Experimental Example 4.
  • the present invention relates to a DNA breakage repair-related protein or a nucleic acid encoding the protein, which has a higher expression level in a cell of a marmoset than a cell of a species having a lower homologous recombination efficiency than a cell of a marmoset.
  • homologous recombination efficiency (example repaired by HDR) / ⁇ (example repaired by NHEJ) + (example repaired by HDR) ⁇ (1)
  • Marmosets (Callithrix jacchus) are a kind of new world monkeys classified as primates, and their body type is small, and their generation change time is as short as about one year, so they are regarded as primate model animals.
  • the inventors have found that the homologous recombination efficiency is less than 1% in human ES cells (embryonic stem cells) and iPS cells (inducible pluripotent stem cells) and 1% in mouse ES cells. It was found to be higher than 80% in marmoset ES cells compared to ⁇ 5%.
  • the cells to be compared for the efficiency of homologous recombination with marmosets may be, for example, pluripotent stem cells, differentiated cells, or established cells such as cancer cells. Is also good. Pluripotent stem cells include tissue stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like.
  • Species having low homologous recombination efficiency include, but are not limited to, humans, mice, rats, rabbits, monkeys, pigs, goats, sheep, cows, ferrets, dogs, cats, chickens, and the like.
  • RNA sequencing RNA-Seq
  • microarray microarray
  • electrophoresis two-dimensional electrophoresis
  • DNA break repair-related proteins include RAD51 paralog, homologous recombination factor involved in susceptibility to breast cancer, and the like.
  • the protein may be a human protein, a marmoset protein, or a protein of another animal species.
  • RAD51 paralog examples include RAD51B, RAD51C, RAD51D, XRCC2 (RefSeq @ ID @ NP_005422.1), and XRCC3 (RefSeq @ ID @ NP_0010935888.1).
  • Examples of homologous recombination factors related to breast cancer susceptibility include ATM (RefSeq @ ID @ NP_000004.3), BARD1 (RefSeq @ ID @ NP_0004566.2), BRIP1 (RefSeq @ ID @ NP_114432.2), CHEK2 ([email protected]). (RefSeq @ ID @ NP_001317276.1), MSH6 (RefSeq @ ID @ NP_0001700.1), NBN (RefSeq @ ID @ NP_001019859.1), PALB2 (RefSeq @ ID @ NP_07895.12), PMS2 (RefSeqA ID50N2). 2) and the like, but are not limited thereto. .
  • ATM RefSeq @ ID @ NP_000004.3
  • BARD1 RefSeq @ ID @ NP_0004566.2
  • BRIP1 RefSeq @ ID @ NP
  • the DNA cleavage repair-related protein one of the above-mentioned proteins may be used alone, or two or more may be used in combination. Further, the homologous recombination efficiency increasing agent of the present embodiment is not limited to a protein, and may be RNA or DNA encoding the protein.
  • the DNA break repair-related protein may be RAD51C, RAD51D, BRCA1, BRCA2.
  • BRCA1 and BRCA2 may be used as a mixture
  • RAD51C and RAD51D may be used as a mixture
  • BRCA1, BRCA2, RAD51C and RAD51D may be used as a mixture
  • BRCA2, RAD51C, RAD51D and RAD51 may be used as a mixture.
  • Tables 1 and 2 show examples of BRCA1, BRCA2, RAD51C and RAD51D proteins of human, marmoset, and mouse, and NCBI accession numbers of mRNAs encoding the proteins.
  • the agent for increasing the efficiency of homologous recombination of the present embodiment is not limited to the DNA break repair-related proteins exemplified in Tables 1 and 2 and mutants of the nucleic acids encoding them, as long as they have the activity of increasing the efficiency of homologous recombination. Good.
  • homologous recombination efficiency increasing agent is a protein or RNA
  • homologous recombination efficiency can be increased by incorporating these into cells by a standard method such as microinjection or electroporation.
  • the homologous recombination efficiency increasing agent may be an expression construct in which a DNA encoding the above-mentioned DNA cleavage repair-related protein is linked to a promoter.
  • the promoter may be one having activity in a target cell or an expression-inducible promoter capable of inducing the activity by a drug or the like.
  • the promoter may be, for example, a cytomegalovirus promoter (CMV promoter) or a CMV early enhancer / chicken beta actin (CAG promoter) having strong promoter activity in almost all cells, or a tissue-specific promoter.
  • CMV promoter cytomegalovirus promoter
  • CAG promoter CMV early enhancer / chicken beta actin
  • a promoter having promoter activity may be used.
  • expression-inducible promoter examples include a doxycycline-inducible promoter (TetO promoter) which can artificially control the promoter activity.
  • TetO promoter doxycycline-inducible promoter
  • the expression construct may be, for example, one integrated into a transposon vector, an adenovirus vector, an adeno-associated virus vector, a plasmid vector, an episomal vector, or the like.
  • a vector that is not integrated into the genome or a vector that is temporarily integrated into the genome but can be removed from the genome by an artificial operation is preferable.
  • Examples of a method for introducing a vector into a cell include a lipofection method, a DEAE-dextran method, a calcium phosphate method, a microinjection method, an electroporation method, and a virus vector method.
  • the present invention relates to a method for producing a cell having an increased homologous recombination efficiency, wherein the cell is a species of a cell having a lower homologous recombination efficiency as compared to a marmoset cell,
  • a production method is provided, which comprises introducing a replacement efficiency increasing agent.
  • the species having a low homologous recombination efficiency as compared with the marmoset cells include, for example, human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat, and chicken. It is selected from a group, but is not limited to this.
  • the present invention provides a species cell having a lower homologous recombination efficiency than a marmoset cell, into which the agent for increasing homologous recombination efficiency is introduced.
  • the cells of the present embodiment have an increased homologous recombination efficiency as compared with the case where the homologous recombination efficiency increasing agent is not introduced.
  • the species having a low homologous recombination efficiency as compared with the marmoset cells include, for example, human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat, and chicken. It is selected from a group, but is not limited to this.
  • the present invention relates to a method for producing a cell in which genomic DNA has been modified by homologous recombination, wherein the cell is a cell of a species having a lower homologous recombination efficiency than a cell of a marmoset,
  • the present invention provides a production method, which comprises introducing the above-mentioned agent for increasing the efficiency of homologous recombination into a cell, introducing a donor DNA into the cell, and cutting the genomic DNA of the cell.
  • the species having a low homologous recombination efficiency as compared to cells of a marmoset are, for example, human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. Selected from, but not limited to.
  • the donor DNA may be a single-stranded DNA of about 50 to 5,000 base pairs or a double-stranded DNA of about 500 to 5,000 base pairs. It is preferable that the donor DNA has 90% or more, preferably 95% or more, more preferably 99% or more sequence identity with the region including the position of the double-stranded DNA break of the genomic DNA.
  • sequence identity is a value indicating a ratio of a target base sequence to a reference base sequence (reference base sequence).
  • the donor DNA may include, for example, a gene encoding a fluorescent protein or a protein tag.
  • the donor DNA may be designed by HDR such that the gene located near the genomic DNA break and the gene express a fusion protein.
  • genomic DNA breaks in cells is very low.
  • the genomic DNA can be specifically cleaved by causing cells to express an enzyme that cleaves the genomic DNA artificially.
  • the enzyme is not particularly limited as long as it cleaves genomic DNA in a target sequence-specific manner.
  • the length of the target sequence recognized by the enzyme that cuts genomic DNA may be, for example, about 10 to 40 bases.
  • the enzyme that cleaves genomic DNA may be an RNA-induced artificial nuclease or an artificial nuclease generally used in so-called genome editing.
  • RNA-induced artificial nuclease is an enzyme in which short RNA serving as a guide binds to a target sequence and nuclease cleaves DNA specifically in the target sequence.
  • RNA-induced artificial nucleases include, but are not limited to, Cas family proteins.
  • Artificial nuclease is an artificial restriction enzyme obtained by fusing a DNA binding domain that binds to a target sequence with the nuclease domain of restriction enzyme FokI.
  • Artificial nucleases include, but are not limited to, Zinc finger nuclease (ZFN), Transcription activator-like effector nuclease (TALEN), and the like.
  • nickase may be used as an enzyme that cuts genomic DNA.
  • Nickase is an enzyme that cuts one strand of double-stranded DNA. For example, at close locations on genomic DNA, a nickase nicks both strands of the double-stranded DNA, thereby forming a double-stranded DNA break.
  • BRCA1, BRCA2, RAD51C and RAD51D are more highly expressed in marmoset ES cells than in human ES / iPS cells.
  • DSY127 indicates the cell line name of marmoset embryonic stem cells.
  • 201B7, WD39 and etKA4 are iPS cell lines, and human iPSKhES-1 is a human ES cell line.
  • FIG. 2 shows the result of comparing the expression level of the RAD51C gene
  • FIG. 3 shows the result of comparing the expression level of the RAD51D gene
  • FIG. 4 shows the result of comparing the expression level of the RAD51 gene
  • FIG. Shows the results of comparing the expression levels of the BRCA1 gene
  • FIG. 6 shows the results of comparing the expression levels of the BRCA2 gene.
  • HPRT WT indicates a normal human HPRT locus
  • HPRT targeting vector indicates a targeting vector containing a neomycin resistance gene
  • HPRT Neo indicates an HPRT locus after homologous recombination.
  • 6-thioguanine 6-thioguanine
  • gentamicin analog G418 inhibits eukaryotic protein synthesis and kills cells, cells transformed with the neomycin resistance gene inserted can survive in the presence of G418.
  • Cas9 protein was used as an enzyme for cleaving genomic DNA.
  • As the sequence of the guide RNA one targeting exon 2 of the HPRT gene was used.
  • the nucleotide sequence of the target sequence of the guide RNA is shown in SEQ ID NO: 1.
  • a donor DNA having a sequence homologous to the HPRT locus and a neomycin resistance gene cassette was prepared.
  • the nucleotide sequence of the donor DNA is shown in SEQ ID NO: 2.
  • As the donor DNA a circular double-stranded DNA was used.
  • the cells were divided into two equal groups, one of which was supplemented with G418 alone and the other of which was supplemented with G418 and 6-TG, cultured and sorted. The number of colonies in each of the two groups was counted.
  • the number of G418-resistant clones in which the neomycin resistance gene cassette was inserted at any position on the genome reflects the total number of homologous recombinants and non-homologous recombinants.
  • the number of clones resistant to both G418 and 6-TG in which the neomycin gene cassette was inserted at the HPRT locus reflects the number of homologous recombinants.
  • the homologous recombination efficiency can be calculated by calculating the ratio of the number of colonies of cells resistant to both G418 and 6-TG to the number of colonies of G418-resistant cells from the following equation (3).
  • (Homologous recombination efficiency) (number of colonies of cells resistant to both G418 and 6-TG) / (number of colonies of G418-resistant cells) (3)
  • the NCBI accession number of the RAD51 mRNA is NM — 0028755.4, and the NCBI accession number of the RAD protein is NP — 0028666.2.
  • the NCBI accession numbers of BRCA1, BRCA2, RAD51C and RAD51D are as shown in Tables 1 and 2.
  • the expression constructs of the plurality of DNA-cutting repair-related proteins are mixed with the donor DNA construct and the expression construct of an enzyme that cuts genomic DNA, and introduced into human iPS cells. did.
  • the cells were divided into two equal groups, one of which was supplemented with G418 alone and the other of which was supplemented with G418 and 6-TG, cultured and sorted. The number of colonies in each of the two groups was counted.
  • Homologous recombination efficiency was calculated from the measured number of colonies using the above formula (3). The results are shown in FIG. 7 and FIG. The vertical axis in FIGS. 7 and 8 indicates the homologous recombination efficiency calculated by the above equation (3).
  • FIG. 7 shows the homologous recombination efficiency when one or two or more DNA break repair-related proteins are expressed. In FIGS. 7 and 8, mock shows the case where the DNA break repair-related protein was not expressed.
  • the homologous recombination efficiency is significantly increased when the DNA break repair-related proteins are expressed in combination, that is, when BRCA1 and BRCA2, RAD51C and RAD51D, and BRCA1 and BRCA2, RAD51C and RAD51D are expressed. It turned out to be.
  • Wild type indicates the Sox2 locus on the genomic DNA
  • Knock-in indicates the Sox2 locus after homologous recombination in which Venus was inserted in-frame at the Sox2 locus.
  • the Cas9 protein was used as an enzyme that cuts genomic DNA.
  • the guide RNA was designed to target around the stop codon of the SOX2 gene.
  • the EB3 strain was used as mouse ES cells.
  • a donor DNA having an ORF encoding Venus, about 500 base pairs upstream of the stop codon of the SOX2 gene, and about 500 base pairs downstream of the stop codon of the SOX2 gene was prepared.
  • the Venus ORF designed the donor DNA to ligate in frame with the SOX2 gene.
  • As the donor DNA a circular double-stranded DNA was used.
  • an expression construct for a DNA cleavage repair-related protein an expression construct in which a human BRCA1 gene, a human BRCA2 gene, a human RAD51C gene, a human RAD51D gene, and a human RAD51 gene were respectively linked to the downstream of a CAG promoter was used.
  • a mixture of an expression construct for a DNA cleavage repair-related protein, a donor DNA, and an expression construct for Cas9 protein and guide RNA was introduced into mouse ES cells by electroporation. Subsequently, the obtained cells were seeded and cultured. The results are shown in FIG.
  • FIG. 10 is a micrograph of cells expressing Venus after homologous recombination. As a result, it was revealed that in cells in which Venus was inserted into the Sox2 locus by homologous recombination, the SOX2 :: Venus fusion protein was localized in the nucleus.
  • the cells subjected to electroporation were cultured to form colonies. Among the formed colonies, the ratio of colonies having Venus-expressing cells (Venus positive colony ratio) was measured. The results are shown in FIG.
  • FIG. 11 shows the results showing the ratio of Venus-positive colonies in the case of expressing the DNA cleavage repair-related protein.
  • 4F shows the case where BRCA1, BRCA2, RAD51C and RAD51D are simultaneously expressed
  • 5F shows the case where BRCA1, BRCA2, RAD51C, RAD51D and RAD51 are simultaneously expressed.
  • Mock shows the case where the DNA break repair-related protein was not expressed.
  • a technique for improving the efficiency of homologous recombination can be provided.

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Abstract

A homologous recombination efficiency increasing agent containing a DNA break repair-related protein or a nucleic acid encoding the protein as an active ingredient, wherein the DNA break repair-related protein can be expressed in a larger amount in a cell in marmoset compared with a cell in a species in which the homologous recombination efficiency is lower compared with that in a cell in marmoset.

Description

相同組換え効率上昇剤及びその使用Agent for increasing homologous recombination efficiency and use thereof
　本発明は、相同組換え効率上昇剤及びその使用に関する。より詳細には、相同組換え効率上昇剤、相同組換え効率が上昇した細胞の製造方法及び相同組換えによりゲノムＤＮＡが改変された細胞の製造方法に関する。本願は、２０１８年９月５日に日本に出願された特願２０１８－１６５８１３号に基づき優先権を主張し、それらの内容をここに援用する。 (4) The present invention relates to an agent for increasing the efficiency of homologous recombination and its use. More specifically, the present invention relates to an agent for increasing the efficiency of homologous recombination, a method for producing a cell having an increased efficiency of homologous recombination, and a method for producing a cell having genomic DNA modified by homologous recombination. This application claims priority based on Japanese Patent Application No. 2018-165813 for which it applied to Japan on September 5, 2018, and uses those content here.
　相同組換えとは、異なるふたつの二本鎖ＤＮＡのうち一方の二本鎖ＤＮＡが切断され、その切断箇所の近傍に相同なＤＮＡ配列が存在する場合に、その相同配列において交叉が起き、前記二本鎖ＤＮＡの間でＤＮＡ配列が交換されることを指す。 Homologous recombination means that when one double-stranded DNA of two different double-stranded DNAs is cleaved and a homologous DNA sequence exists near the cleavage site, crossover occurs in the homologous sequence, Refers to the exchange of DNA sequences between double-stranded DNA.
　部位特異的ヌクレアーゼを用いて任意のゲノム領域にＤＮＡ二本鎖切断を引き起こし、ＤＮＡ修復機構を利用する遺伝子改変技術はゲノム編集と呼ばれ、細胞あるいは個体レベルでの遺伝子ノックイン・ノックアウトが可能である。 Genetic modification technology that uses a site-specific nuclease to cause DNA double-strand breaks in any genomic region and uses the DNA repair mechanism is called genome editing, and gene knock-in / knock-out at the cell or individual level is possible. .
　例えば、非特許文献１には、ゲノム編集技術を用いて、マウスＥＳ細胞において、５種の遺伝子を同時に変異させた例が示されている。 For example, Non-Patent Document 1 shows an example in which five genes are simultaneously mutated in mouse ES cells using genome editing technology.
　ＤＮＡ修復機構には２種類あり、一つは非相同末端結合（Ｎｏｎ－ｈｏｍｏｌｏｇｏｕｓ　ｅｎｄ　ｊｏｉｎｉｎｇ、ＮＨＥＪ）である。この経路を用いたゲノム編集は、切断末端の修復過程において頻繁に起こる塩基の欠失・挿入等のエラーによるフレームシフトを利用し、遺伝子機能の喪失（ノックアウト）を起こすものである。 There are two types of DNA repair mechanisms, one of which is non-homologous end joining (NHEJ). Genome editing using this pathway utilizes a frame shift due to an error such as deletion or insertion of a base that frequently occurs in the process of repairing a cleaved end to cause loss of gene function (knockout).
　もう一つは相同指向性修復（Ｈｏｍｏｌｏｇｙ－ｄｉｒｅｃｔｅｄ　ＤＮＡ　ｒｅｐａｉｒ、ＨＤＲ）であり、部位特異的ＤＮＡ二本鎖切断の際に、標的部位と相同な配列を持つ１本鎖オリゴＤＮＡあるいは二本鎖ＤＮＡ等のドナーＤＮＡを加えることで、相同組換えによって任意の変異あるいはＤＮＡ断片を標的部位に挿入（ノックイン）することが可能である。 The other is homology-directed DNA repair (HDR), which is a single-stranded oligo DNA or double-stranded DNA having a sequence homologous to a target site upon site-specific DNA double-strand breaks. By adding a donor DNA such as described above, it is possible to insert (knock-in) an arbitrary mutation or DNA fragment into a target site by homologous recombination.
　多くの遺伝性疾患においては、１塩基置換等の変異により引き起こされることが多いため、ヒト疾患ｉＰＳ細胞や疾患モデルマウスの作製において、ノックイン技術はノックアウトと比較して優位性を持つ。また、時空間特異的なコンディショナル・ノックアウトなど高度な遺伝子改変を行う際にもノックイン技術は欠かせないものとなる。 た め Since many genetic diseases are often caused by mutations such as single nucleotide substitutions, the knock-in technique has an advantage over the knock-out technique in the production of human disease iPS cells and disease model mice. Knock-in technology is also indispensable for advanced genetic modification such as spatio-temporal conditional knockout.
　ＮＨＥＪを介したＤＮＡ修復は、挿入・欠失させるＤＮＡ配列を制御することができない。標的部位に所望のＤＮＡ断片を挿入させるためには、ＨＤＲを介したＤＮＡ修復を利用する必要がある。 修復 DNA repair via NHEJ cannot control the DNA sequence to be inserted or deleted. In order to insert a desired DNA fragment into a target site, it is necessary to use HDR-mediated DNA repair.
　しかしながら、ＨＤＲによるＤＮＡ修復はＮＨＥＪよりも頻度が非常に低いため、一般にノックインの効率は単純ノックアウトよりもはるかに劣る。そこで、本発明では、相同組換え効率を改善する技術を提供することを目的とする。 However, DNA repair by HDR is much less frequent than NHEJ, so knock-in efficiency is generally much lower than simple knock-out. Therefore, an object of the present invention is to provide a technique for improving the efficiency of homologous recombination.
　本発明は、以下の態様を含む。
［１］マーモセットの細胞と比較して相同組換え効率が低い種の細胞よりも、マーモセットの細胞における発現量が高い、ＤＮＡ切断修復関連タンパク質又は前記タンパク質をコードする核酸を有効成分として含有する、相同組換え効率上昇剤。
［２］マーモセットの細胞と比較して相同組換え効率が低い前記種が、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択される、請求項１に記載の相同組換え効率上昇剤。
［３］前記ＤＮＡ切断修復関連タンパク質が、ＲＡＤ５１パラログ及び乳癌易罹患性に関わる相同組換え因子である［１］または［２］に記載の相同組換え効率上昇剤。
［４］前記ＤＮＡ切断修復関連タンパク質が、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄからなる群より選択される、［３］に記載の相同組換え効率上昇剤。
［５］相同組換え効率が上昇した細胞の製造方法であって、前記細胞はマーモセットの細胞と比較して相同組換え効率が低い種の細胞であり、前記細胞に［１］～［４］のいずれか一項に記載の相同組換え効率上昇剤を導入することを含む、製造方法。
［６］マーモセットの細胞と比較して相同組換え効率が低い前記種が、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択される、［５］に記載の製造方法。
［７］［１］～［４］のいずれか一項に記載の相同組換え効率上昇剤が導入された、マーモセットの細胞と比較して相同組換え効率が低い種の細胞。
［８］マーモセットの細胞と比較して相同組換え効率が低い前記種が、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択される、［７］に記載の細胞。
［９］相同組換えによりゲノムＤＮＡが改変された細胞の製造方法であって、前記細胞はマーモセットの細胞と比較して相同組換え効率が低い種の細胞であり、前記細胞に［１］～［４］のいずれか一項に記載の相同組換え効率上昇剤を導入することと、前記細胞にドナーＤＮＡを導入することと、前記細胞のゲノムＤＮＡを切断することと、を含む、製造方法。
［１０］マーモセットの細胞と比較して相同組換え効率が低い前記種が、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択される、［９］に記載の製造方法。 The present invention includes the following aspects.
[1] containing, as an active ingredient, a DNA cleavage repair-related protein or a nucleic acid encoding the protein, which has a higher expression level in marmoset cells than a cell of a species having a lower homologous recombination efficiency than a marmoset cell; Agent for increasing homologous recombination efficiency.
[2] The species whose homologous recombination efficiency is lower than that of marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. The agent for increasing the efficiency of homologous recombination according to claim 1, which is used.
[3] The agent for increasing homologous recombination efficiency according to [1] or [2], wherein the DNA breakage repair-related protein is a RAD51 paralog and a homologous recombination factor involved in susceptibility to breast cancer.
[4] The agent for increasing homologous recombination efficiency according to [3], wherein the DNA breakage repair-related protein is selected from the group consisting of BRCA1, BRCA2, RAD51C, and RAD51D.
[5] A method for producing a cell having an increased homologous recombination efficiency, wherein the cell is a species of a cell having a lower homologous recombination efficiency as compared to a marmoset cell, and the cells have [1] to [4]. A production method comprising introducing the homologous recombination efficiency increasing agent according to any one of the above.
[6] The species whose homologous recombination efficiency is lower than that of marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. Production method according to [5].
[7] A cell of a species having a lower homologous recombination efficiency than a marmoset cell, into which the agent for increasing a homologous recombination efficiency according to any one of [1] to [4] is introduced.
[8] The species whose homologous recombination efficiency is lower than that of marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat, and chicken. The cell according to [7], which is obtained.
[9] A method for producing a cell in which genomic DNA has been modified by homologous recombination, wherein the cell is a species of a cell having a lower homologous recombination efficiency than a marmoset cell, and [4] A production method, comprising: introducing the homologous recombination efficiency increasing agent according to any one of [4], introducing a donor DNA into the cell, and cutting the genomic DNA of the cell. .
[10] The species having a lower homologous recombination efficiency as compared to marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat, and chicken. Production method according to [9].
　本発明によれば、相同組換え効率を改善する技術を提供することができる。 According to the present invention, a technique for improving the efficiency of homologous recombination can be provided.
実験例２における、ＨＰＲＴ遺伝子座において相同組換えを引き起こし、相同組換え効率を算出する実験系について説明する図である。FIG. 8 is a diagram illustrating an experimental system in Experimental Example 2 for causing homologous recombination at the HPRT locus and calculating homologous recombination efficiency. 実験例２における、マーモセットＥＳ細胞又はヒトｉＰＳ／ＥＳ細胞においての、ＲＡＤ５１Ｃ遺伝子の発現をリアルタイムＰＣＲによって測定した結果である。FIG. 9 shows the results of measuring the expression of the RAD51C gene in marmoset ES cells or human iPS / ES cells by real-time PCR in Experimental Example 2. FIG. 実験例２における、マーモセットＥＳ細胞又はヒトｉＰＳ／ＥＳ細胞においての、ＲＡＤ５１Ｄ遺伝子の発現をリアルタイムＰＣＲによって測定した結果である。It is the result of having measured the expression of the RAD51D gene in the marmoset ES cell or the human iPS / ES cell in Experimental Example 2 by real-time PCR. 実験例２における、マーモセットＥＳ細胞又はヒトｉＰＳ／ＥＳ細胞においての、ＲＡＤ５１遺伝子の発現をリアルタイムＰＣＲによって測定した結果である。5 shows the results of measuring the expression of the RAD51 gene in marmoset ES cells or human iPS / ES cells in real time PCR in Experimental Example 2. 実験例２における、マーモセットＥＳ細胞又はヒトｉＰＳ／ＥＳ細胞においての、ＢＲＣＡ１遺伝子の発現をリアルタイムＰＣＲによって測定した結果である。5 shows the results of real-time PCR measuring the expression of the BRCA1 gene in marmoset ES cells or human iPS / ES cells in Experimental Example 2. 実験例２における、マーモセットＥＳ細胞又はヒトｉＰＳ／ＥＳ細胞においての、ＢＲＣＡ２遺伝子の発現をリアルタイムＰＣＲによって測定した結果である。5 shows the results of measuring the expression of the BRCA2 gene in marmoset ES cells or human iPS / ES cells in real time PCR in Experimental Example 2. 実験例３における、ヒトｉＰＳ細胞に各ＤＮＡ切断修復関連タンパク質を発現させた時の、ＨＰＲＴ遺伝子における相同組換え効率を算出した結果である。FIG. 9 shows the results of calculating the homologous recombination efficiency of the HPRT gene when each DNA breakage repair-related protein was expressed in human iPS cells in Experimental Example 3. FIG. 実験例３における、ヒトｉＰＳ細胞に各ＤＮＡ切断修復関連タンパク質（ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄ）を組み合わせて発現させた時の、ＨＰＲＴ遺伝子における相同組換え効率を算出した結果である。FIG. 10 shows the results of calculating the homologous recombination efficiency in the HPRT gene in Experimental Example 3 when human iPS cells were expressed in combination with DNA breakage repair-related proteins (BRCA1, BRCA2, RAD51C, and RAD51D). 実験例４における、Ｓｏｘ２遺伝子座において相同組換えを行う実験系について説明する図である。FIG. 9 is a diagram illustrating an experimental system for performing homologous recombination at the Sox2 locus in Experimental Example 4. 実験例４における、核においてＳＯＸ２：：Ｖｅｎｕｓ融合タンパク質を発現する細胞の顕微鏡像である。9 is a microscope image of cells expressing SOX2 :: Venus fusion protein in the nucleus in Experimental Example 4. 実験例４における、Ｖｅｎｕｓ陽性コロニー率を算出した結果である。9 shows the results of calculating the Venus-positive colony rate in Experimental Example 4.
［相同組換え効率上昇剤］
　１実施形態において、本発明は、マーモセットの細胞と比較して相同組換え効率が低い種の細胞よりも、マーモセットの細胞における発現量が高い、ＤＮＡ切断修復関連タンパク質又は前記タンパク質をコードする核酸を有効成分として含有する、相同組換え効率上昇剤を提供する。 [Homologous recombination efficiency increasing agent]
In one embodiment, the present invention relates to a DNA breakage repair-related protein or a nucleic acid encoding the protein, which has a higher expression level in a cell of a marmoset than a cell of a species having a lower homologous recombination efficiency than a cell of a marmoset. An agent for increasing the efficiency of homologous recombination, which is contained as an active ingredient.
　本明細書において、相同組換え効率は、下記式（１）にしたがって求めることができる。
（相同組換え効率）＝（ＨＤＲにより修復された例）／｛（ＮＨＥＪにより修復された例）＋（ＨＤＲにより修復された例）｝　（１） In the present specification, the homologous recombination efficiency can be determined according to the following formula (1).
(Homologous recombination efficiency) = (example repaired by HDR) / {(example repaired by NHEJ) + (example repaired by HDR)} (1)
　マーモセット（Ｃａｌｌｉｔｈｒｉｘ　ｊａｃｃｈｕｓ）は、霊長目に分類される新世界ザルの一種であり、その体型は小型で、世代交代時間が約１年と短いことから、霊長類のモデル動物として位置づけられている。 Marmosets (Callithrix jacchus) are a kind of new world monkeys classified as primates, and their body type is small, and their generation change time is as short as about one year, so they are regarded as primate model animals.
　実施例において詳述するように、発明者らは、相同組換え効率は、ヒトＥＳ細胞（胚性幹細胞）及びｉＰＳ細胞（人工多能性幹細胞）では１％未満であり、マウスＥＳ細胞では１～５％であるのに対し、マーモセットＥＳ細胞では８０％より高いことを見出した。 As described in detail in the examples, the inventors have found that the homologous recombination efficiency is less than 1% in human ES cells (embryonic stem cells) and iPS cells (inducible pluripotent stem cells) and 1% in mouse ES cells. It was found to be higher than 80% in marmoset ES cells compared to ～ 5%.
　マーモセットと相同組換え効率を比較する対象の細胞としては、例えば、多能性幹細胞であってもよいし、分化した細胞であってもよいし、癌細胞等の株化した細胞等であってもよい。多能性幹細胞としては、組織幹細胞、胚性幹細胞（ＥＳ細胞）及び人工多能性幹細胞（ｉＰＳ細胞）等が挙げられる。 The cells to be compared for the efficiency of homologous recombination with marmosets may be, for example, pluripotent stem cells, differentiated cells, or established cells such as cancer cells. Is also good. Pluripotent stem cells include tissue stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like.
　相同組換え効率が低い種としては、例えば、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリ等が挙げられるが、これに限定されない。 種 Species having low homologous recombination efficiency include, but are not limited to, humans, mice, rats, rabbits, monkeys, pigs, goats, sheep, cows, ferrets, dogs, cats, chickens, and the like.
　発現量を比較する方法としては、例えば、ＲＮＡシーケンシング（ＲＮＡ－Ｓｅｑ）、マイクロアレイ、二次元電気泳動などが挙げられるが、これに限定されない。 方法 Methods for comparing the expression levels include, but are not limited to, RNA sequencing (RNA-Seq), microarray, two-dimensional electrophoresis, and the like.
　ＤＮＡ切断修復関連タンパク質としては、例えば、ＲＡＤ５１パラログ、乳癌易罹患性に関わる相同組換え因子等が挙げられる。前記タンパク質はヒトタンパク質であってもよいし、マーモセットタンパク質であってもよいし、他動物種のタンパク質であってもよい。 Examples of DNA break repair-related proteins include RAD51 paralog, homologous recombination factor involved in susceptibility to breast cancer, and the like. The protein may be a human protein, a marmoset protein, or a protein of another animal species.
　ＲＡＤ５１パラログとしては、ＲＡＤ５１Ｂ、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄ、ＸＲＣＣ２（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿００５４２２．１）、ＸＲＣＣ３（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿００１０９３５８８．１）等が挙げられる。 Examples of the RAD51 paralog include RAD51B, RAD51C, RAD51D, XRCC2 (RefSeq @ ID @ NP_005422.1), and XRCC3 (RefSeq @ ID @ NP_0010935888.1).
　乳癌易罹患性に関わる相同組換え因子としては、ＡＴＭ（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿００００４２．３）、ＢＡＲＤ１（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿０００４５６．２）、ＢＲＩＰ１（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿１１４４３２．２）、ＣＨＥＫ２（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿００１００５７３５．１）、ＭＲＥ１１Ａ（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿００１３１７２７６．１）、ＭＳＨ６（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿０００１７０．１）、ＮＢＮ（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿００１０１９８５９．１）、ＰＡＬＢ２（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿０７８９５１．２）、ＰＭＳ２（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿０００５２６．２）、ＲＡＤ５０（ＲｅｆＳｅｑ　ＩＤ　ＮＰ＿００５７２３．２）等が挙げられるが、これに限定されない。 Examples of homologous recombination factors related to breast cancer susceptibility include ATM (RefSeq @ ID @ NP_000004.3), BARD1 (RefSeq @ ID @ NP_0004566.2), BRIP1 (RefSeq @ ID @ NP_114432.2), CHEK2 ([email protected]). (RefSeq @ ID @ NP_001317276.1), MSH6 (RefSeq @ ID @ NP_0001700.1), NBN (RefSeq @ ID @ NP_001019859.1), PALB2 (RefSeq @ ID @ NP_07895.12), PMS2 (RefSeqA ID50N2). 2) and the like, but are not limited thereto. .
　また、ＤＮＡ切断修復関連タンパク質としては、上述したもののうち一種類を単独で用いてもよいし、二種類以上を混合して用いてもよい。さらに、本実施形態の相同組換え効率上昇剤は、タンパク質に限定されず、そのタンパク質をコードするＲＮＡやＤＮＡであってもよい。 As the DNA cleavage repair-related protein, one of the above-mentioned proteins may be used alone, or two or more may be used in combination. Further, the homologous recombination efficiency increasing agent of the present embodiment is not limited to a protein, and may be RNA or DNA encoding the protein.
　ＤＮＡ切断修復関連タンパク質は、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄ、ＢＲＣＡ１、ＢＲＣＡ２であってもよい。
　ＤＮＡ切断修復関連タンパク質としては、ＢＲＣＡ１及びＢＲＣＡ２を混合して用いてもよく、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄを混合して用いてもよく、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄを混合して用いてもよく、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄ及びＲＡＤ５１を混合して用いてもよい。 The DNA break repair-related protein may be RAD51C, RAD51D, BRCA1, BRCA2.
As the DNA cleavage repair-related protein, BRCA1 and BRCA2 may be used as a mixture, RAD51C and RAD51D may be used as a mixture, and BRCA1, BRCA2, RAD51C and RAD51D may be used as a mixture. BRCA2, RAD51C, RAD51D and RAD51 may be used as a mixture.
　ヒト、マーモセット、マウスのＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄタンパク質、及び、そのタンパク質をコードするｍＲＮＡのＮＣＢＩアクセッション番号の例を表１及び２に示す。 Tables 1 and 2 show examples of BRCA1, BRCA2, RAD51C and RAD51D proteins of human, marmoset, and mouse, and NCBI accession numbers of mRNAs encoding the proteins.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　本実施形態の相同組換え効率上昇剤は、相同組換え効率を上昇させる活性を持つ限り、表１と表２に例示したＤＮＡ切断修復関連タンパク質及びそれらをコードする核酸の変異体であってもよい。 The agent for increasing the efficiency of homologous recombination of the present embodiment is not limited to the DNA break repair-related proteins exemplified in Tables 1 and 2 and mutants of the nucleic acids encoding them, as long as they have the activity of increasing the efficiency of homologous recombination. Good.
　相同組換え効率上昇剤がタンパク質又はＲＮＡである場合には、マイクロインジェクション、エレクトロポレーション等の定法によりこれらを細胞に取り込ませることにより、相同組換え効率を上昇させることができる。 (4) When the homologous recombination efficiency increasing agent is a protein or RNA, homologous recombination efficiency can be increased by incorporating these into cells by a standard method such as microinjection or electroporation.
　相同組換え効率上昇剤は、上記ＤＮＡ切断修復関連タンパク質をコードするＤＮＡとプロモーターを連結した発現コンストラクトであってもよい。プロモーターとしては、対象細胞において活性を有するものであってもよいし、薬剤などにより活性を誘導可能な発現誘導型プロモーターであってもよい。 (4) The homologous recombination efficiency increasing agent may be an expression construct in which a DNA encoding the above-mentioned DNA cleavage repair-related protein is linked to a promoter. The promoter may be one having activity in a target cell or an expression-inducible promoter capable of inducing the activity by a drug or the like.
　プロモーターとしては、例えば、ほぼ全ての細胞において強いプロモーター活性を有する、サイトメガロウイルス・プロモーター（ＣＭＶプロモーター）やＣＭＶ　ｅａｒｌｙ　ｅｎｈａｎｃｅｒ／ｃｈｉｃｋｅｎ　ｂｅｔａ　ａｃｔｉｎ（ＣＡＧプロモーター）等であってもよいし、組織特異的なプロモーター活性を有するプロモーター等であってもよい。 The promoter may be, for example, a cytomegalovirus promoter (CMV promoter) or a CMV early enhancer / chicken beta actin (CAG promoter) having strong promoter activity in almost all cells, or a tissue-specific promoter. A promoter having promoter activity may be used.
　発現誘導型プロモーターとしては、人為的にプロモーター活性を制御することのできる、ドキシサイクリン誘導型プロモーター（ＴｅｔＯプロモーター）等が挙げられる。 Examples of the expression-inducible promoter include a doxycycline-inducible promoter (TetO promoter) which can artificially control the promoter activity.
　前記発現コンストラクトは、例えば、トランスポゾンベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、プラスミドベクター、エピソーマルベクター等に組み込まれたものであってもよい。 The expression construct may be, for example, one integrated into a transposon vector, an adenovirus vector, an adeno-associated virus vector, a plasmid vector, an episomal vector, or the like.
　ベクターとしては、ベクターがゲノムに組み込まれないもの、又は、一時的にゲノムに組み込まれるが、人為的な操作によってゲノムから除去することのできるベクターが好ましい。 As the vector, a vector that is not integrated into the genome or a vector that is temporarily integrated into the genome but can be removed from the genome by an artificial operation is preferable.
　ベクターを細胞に導入する方法としては、例えば、リポフェクション法、ＤＥＡＥ－デキストラン法、リン酸カルシウム法、マイクロインジェクション法、エレクトロポレーション法、ウイルスベクター法等が挙げられる。 方法 Examples of a method for introducing a vector into a cell include a lipofection method, a DEAE-dextran method, a calcium phosphate method, a microinjection method, an electroporation method, and a virus vector method.
［相同組換え効率が上昇した細胞の製造方法］
　１実施形態において、本発明は、相同組換え効率が上昇した細胞の製造方法であって、前記細胞はマーモセットの細胞と比較して相同組換え効率が低い種の細胞であり、上述した相同組換え効率上昇剤を導入することを含む、製造方法を提供する。 [Method for producing cells with increased homologous recombination efficiency]
In one embodiment, the present invention relates to a method for producing a cell having an increased homologous recombination efficiency, wherein the cell is a species of a cell having a lower homologous recombination efficiency as compared to a marmoset cell, A production method is provided, which comprises introducing a replacement efficiency increasing agent.
　本実施形態において、マーモセットの細胞と比較して相同組換え効率が低い種は、例えば、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択されるが、これに限定されない。 In the present embodiment, the species having a low homologous recombination efficiency as compared with the marmoset cells include, for example, human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat, and chicken. It is selected from a group, but is not limited to this.
［相同組換え効率が上昇した細胞］
　１実施形態において、本発明は、前記の相同組換え効率上昇剤が導入された、マーモセットの細胞と比較して相同組換え効率が低い種の細胞を提供する。本実施形態の細胞は、前記相同組換え効率上昇剤が導入されない場合と比較して、相同組換え効率が上昇している。 [Cells with increased homologous recombination efficiency]
In one embodiment, the present invention provides a species cell having a lower homologous recombination efficiency than a marmoset cell, into which the agent for increasing homologous recombination efficiency is introduced. The cells of the present embodiment have an increased homologous recombination efficiency as compared with the case where the homologous recombination efficiency increasing agent is not introduced.
　本実施形態において、マーモセットの細胞と比較して相同組換え効率が低い種は、例えば、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択されるが、これに限定されない。 In the present embodiment, the species having a low homologous recombination efficiency as compared with the marmoset cells include, for example, human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat, and chicken. It is selected from a group, but is not limited to this.
［相同組換えによりゲノムＤＮＡが改変された細胞の製造方法］
　１実施形態において、本発明は、相同組換えによりゲノムＤＮＡが改変された細胞の製造方法であって、前記細胞はマーモセットの細胞と比較して相同組換え効率が低い種の細胞であり、前記細胞に前記の相同組換え効率上昇剤を導入することと、前記細胞にドナーＤＮＡを導入することと、前記細胞のゲノムＤＮＡを切断することと、を含む、製造方法を提供する。 [Method for producing cells in which genomic DNA has been modified by homologous recombination]
In one embodiment, the present invention relates to a method for producing a cell in which genomic DNA has been modified by homologous recombination, wherein the cell is a cell of a species having a lower homologous recombination efficiency than a cell of a marmoset, The present invention provides a production method, which comprises introducing the above-mentioned agent for increasing the efficiency of homologous recombination into a cell, introducing a donor DNA into the cell, and cutting the genomic DNA of the cell.
　本実施形態において、マーモセットの細胞と比較して相同組換え効率が低い前記種は、例えば、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択されるが、これに限定されない。 In this embodiment, the species having a low homologous recombination efficiency as compared to cells of a marmoset are, for example, human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. Selected from, but not limited to.
　ドナーＤＮＡは、５０～５，０００塩基程度の一本鎖ＤＮＡであってもよいし、５００～５，０００塩基対程度の二本鎖ＤＮＡであってもよい。ドナーＤＮＡは、ゲノムＤＮＡの二本鎖ＤＮＡ切断の位置を含む領域と、９０％以上、好ましくは９５％以上、更に好ましくは９９％以上の配列同一性を有することが好ましい。 The donor DNA may be a single-stranded DNA of about 50 to 5,000 base pairs or a double-stranded DNA of about 500 to 5,000 base pairs. It is preferable that the donor DNA has 90% or more, preferably 95% or more, more preferably 99% or more sequence identity with the region including the position of the double-stranded DNA break of the genomic DNA.
　本明細書において、配列同一性とは、対象の塩基配列が、基準となる塩基配列（基準塩基配列）に対して一致している割合を示す値である。基準塩基配列に対する、対象塩基配列の配列同一性は、例えば次のようにして求めることができる。まず、基準塩基配列及び対象塩基配列をアラインメントする。ここで、各塩基配列には、配列同一性が最大となるようにギャップを含めてもよい。続いて、基準塩基配列及び対象塩基配列において、一致した塩基の塩基数を算出し、下記式（２）にしたがって、配列同一性を求めることができる。
　配列同一性（％）＝一致した塩基数／対象塩基配列の総塩基数×１００　（２） In the present specification, the sequence identity is a value indicating a ratio of a target base sequence to a reference base sequence (reference base sequence). The sequence identity of the target base sequence to the reference base sequence can be determined, for example, as follows. First, the reference base sequence and the target base sequence are aligned. Here, a gap may be included in each base sequence so that sequence identity is maximized. Subsequently, in the reference base sequence and the target base sequence, the number of bases that match is calculated, and sequence identity can be determined according to the following formula (2).
Sequence identity (%) = number of matched bases / total number of bases of target base sequence × 100 (2)
　ドナーＤＮＡは、例えば、蛍光タンパク質やタンパク質タグをコードする遺伝子を含んでいてもよい。ＨＤＲにより、ゲノムＤＮＡ切断の近傍に位置する遺伝子と前記遺伝子が融合タンパク質を発現するように、ドナーＤＮＡを設計してもよい。 The donor DNA may include, for example, a gene encoding a fluorescent protein or a protein tag. The donor DNA may be designed by HDR such that the gene located near the genomic DNA break and the gene express a fusion protein.
　通常の生育環境においては、細胞内でゲノムＤＮＡの切断が起こる頻度は非常に低い。人為的にゲノムＤＮＡを切断する酵素を細胞に発現させることにより、特異的にゲノムＤＮＡ切断を起こすことができる。 に お い て Under normal growth conditions, the frequency of genomic DNA breaks in cells is very low. The genomic DNA can be specifically cleaved by causing cells to express an enzyme that cleaves the genomic DNA artificially.
　前記酵素としては、ゲノムＤＮＡを標的配列特異的に切断するものであれば特に制限されない。ゲノムＤＮＡを切断する酵素が認識する標的配列の長さは、例えば、１０～４０塩基程度であってよい。 The enzyme is not particularly limited as long as it cleaves genomic DNA in a target sequence-specific manner. The length of the target sequence recognized by the enzyme that cuts genomic DNA may be, for example, about 10 to 40 bases.
　ゲノムＤＮＡを切断する酵素は、いわゆるゲノム編集において一般的に用いられる、ＲＮＡ誘導型人工ヌクレアーゼや人工ヌクレアーゼであってもよい。 酵素 The enzyme that cleaves genomic DNA may be an RNA-induced artificial nuclease or an artificial nuclease generally used in so-called genome editing.
　ＲＮＡ誘導型人工ヌクレアーゼとは、ガイドとなる短鎖ＲＮＡが標的配列に結合し、ヌクレアーゼが標的配列特異的にＤＮＡを切断する酵素である。ＲＮＡ誘導型人工ヌクレアーゼとしては、Ｃａｓファミリータンパク質が挙げられるが、これに限定されない。 RNA-induced artificial nuclease is an enzyme in which short RNA serving as a guide binds to a target sequence and nuclease cleaves DNA specifically in the target sequence. RNA-induced artificial nucleases include, but are not limited to, Cas family proteins.
　人工ヌクレアーゼは、標的配列に結合するＤＮＡ結合ドメインと、制限酵素　ＦｏｋＩのヌクレアーゼドメインを融合させた人工制限酵素である。人工ヌクレアーゼとしては、Ｚｉｎｃ　ｆｉｎｇｅｒ　ｎｕｃｌｅａｓｅ（ＺＦＮ）、Ｔｒａｎｓｃｒｉｐｔｉｏｎ　ａｃｔｉｖａｔｏｒ－ｌｉｋｅ　ｅｆｆｅｃｔｏｒ　ｎｕｃｌｅａｓｅ（ＴＡＬＥＮ）等が挙げられるが、これらに限定されない。 Artificial nuclease is an artificial restriction enzyme obtained by fusing a DNA binding domain that binds to a target sequence with the nuclease domain of restriction enzyme FokI. Artificial nucleases include, but are not limited to, Zinc finger nuclease (ZFN), Transcription activator-like effector nuclease (TALEN), and the like.
　また、ゲノムＤＮＡを切断する酵素として、例えばニッカーゼを用いてもよい。ニッカーゼとは、二本鎖ＤＮＡのうちの一本鎖を切断する酵素である。例えば、ゲノムＤＮＡ上の近接した位置において、ニッカーゼが二本鎖ＤＮＡの双方の鎖にニックを形成することにより、二本鎖ＤＮＡ切断が形成される。 ニ Alternatively, for example, nickase may be used as an enzyme that cuts genomic DNA. Nickase is an enzyme that cuts one strand of double-stranded DNA. For example, at close locations on genomic DNA, a nickase nicks both strands of the double-stranded DNA, thereby forming a double-stranded DNA break.
　以下、実施例により本発明を説明するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to the following examples.
［実験例１］
（マーモセットＥＳ細胞の相同組換え効率）
　マーモセットＥＳ細胞の相同組換え効率を解析した。
　マーモセットＥＳ細胞株にＣａｓ９及びｓｇＲＮＡ発現ベクター及びターゲティングベクターをリポフェクション法により導入し、薬剤選別によりクローンを得た。それぞれのクローンを増幅し、ＰＣＲあるいはサザンブロッティングにより遺伝型を判定した。 [Experimental example 1]
(Homologous recombination efficiency of marmoset ES cells)
The homologous recombination efficiency of marmoset ES cells was analyzed.
Cas9 and sgRNA expression vectors and targeting vectors were introduced into the marmoset ES cell line by lipofection, and clones were obtained by drug selection. Each clone was amplified and its genotype was determined by PCR or Southern blotting.
　相同組換え効率は、ヒトＥＳ細胞（胚性幹細胞）及びｉＰＳ細胞（人工多能性幹細胞）では１％未満であり、マウスＥＳ細胞では１～５％であることが知られている。これに対し、マーモセットＥＳ細胞の相同組換え効率は、ヒトＥＳ／ｉＰＳ細胞及びマウスＥＳ細胞の相同組換え効率よりも高いことが明らかとなった。 It is known that the homologous recombination efficiency is less than 1% for human ES cells (embryonic stem cells) and iPS cells (artificial pluripotent stem cells), and 1 to 5% for mouse ES cells. In contrast, it was revealed that the homologous recombination efficiency of marmoset ES cells was higher than that of human ES / iPS cells and mouse ES cells.
　ヒトとマーモセットは近縁種であるにもかかわらず、上述したように、相同組換え効率が大きく異なる。発明者らは、マーモセットにおいては、ヒトに比べて、ＤＮＡ切断修復関連タンパク質が高発現し、このことが高い相同組換え効率に寄与している可能性を想定した。 Although humans and marmosets are closely related species, the homologous recombination efficiencies differ greatly as described above. The inventors assumed that marmosets express DNA breakage repair-related proteins at higher levels than humans, which may contribute to higher homologous recombination efficiency.
［実験例２］
（ＤＮＡ切断修復関連タンパク質の発現解析）
　マーモセットＥＳ細胞の高い相同組換え効率に寄与するタンパク質を同定するために、マーモセットＥＳ細胞とヒトＥＳ／ｉＰＳ細胞から、ＲＮＡを分離し、これらＲＮＡについて、ＲＮＡ－Ｓｅｑにより解析を行った。 [Experimental example 2]
(Expression analysis of DNA break repair-related proteins)
In order to identify proteins that contribute to the high homologous recombination efficiency of marmoset ES cells, RNA was separated from marmoset ES cells and human ES / iPS cells, and these RNAs were analyzed by RNA-Seq.
　その結果、マーモセットＥＳ細胞においては、ヒトＥＳ／ｉＰＳ細胞よりも、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄが高発現していることが明らかになった。 As a result, it was revealed that BRCA1, BRCA2, RAD51C and RAD51D are more highly expressed in marmoset ES cells than in human ES / iPS cells.
　続いて、リアルタイムＰＣＲにより、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄ遺伝子の、マーモセットＥＳ細胞とヒトＥＳ／ｉＰＳ細胞における発現量をより正確に解析した。 Subsequently, the expression levels of BRCA1, BRCA2, RAD51C and RAD51D genes in marmoset ES cells and human ES / iPS cells were more accurately analyzed by real-time PCR.
　マーモセットＥＳ細胞、ヒトｉＰＳ／ＥＳ細胞から、スピンカラムによりＲＮＡを分離し、逆転写酵素によりｃＤＮＡを合成した。検出にはリアルタイムＰＣＲ装置（型式ＶｉｉＡ７、サーモフィッシャー社）を用いた。 ＲＮＡ RNA was separated from marmoset ES cells and human iPS / ES cells by a spin column, and cDNA was synthesized by reverse transcriptase. For detection, a real-time PCR device (Model ViiA7, Thermo Fisher) was used.
　各細胞株における各遺伝子の発現量を評価するために、ＧＡＰＤＨ遺伝子を内部標準とした相対値を算出した。結果を図２～６に示す。図中、ＤＳＹ１２７はマーモセット胚性幹細胞の細胞株名を示す。２０１Ｂ７、ＷＤ３９及びｅｔＫＡ４はｉＰＳ細胞株であり、ヒトｉＰＳＫｈＥＳ－１はヒトＥＳ細胞株である。 相 対 To evaluate the expression level of each gene in each cell line, a relative value was calculated using the GAPDH gene as an internal standard. The results are shown in FIGS. In the figure, DSY127 indicates the cell line name of marmoset embryonic stem cells. 201B7, WD39 and etKA4 are iPS cell lines, and human iPSKhES-1 is a human ES cell line.
　図２は、ＲＡＤ５１Ｃ遺伝子の発現量を比較した結果を示し、図３は、ＲＡＤ５１Ｄ遺伝子の発現量を比較した結果を示し、図４は、ＲＡＤ５１遺伝子の発現量を比較した結果を示し、図５は、ＢＲＣＡ１遺伝子の発現量を比較した結果を示し、図６は、ＢＲＣＡ２遺伝子の発現量を比較した結果を示す。 FIG. 2 shows the result of comparing the expression level of the RAD51C gene, FIG. 3 shows the result of comparing the expression level of the RAD51D gene, FIG. 4 shows the result of comparing the expression level of the RAD51 gene, and FIG. Shows the results of comparing the expression levels of the BRCA1 gene, and FIG. 6 shows the results of comparing the expression levels of the BRCA2 gene.
　さらに、マーモセットＥＳ細胞については、他の複数のマーモセットＥＳ細胞においてもＤＳＹ１２７細胞と同様の結果が得られている。その結果、マーモセットＥＳ細胞において、ＲＡＤ５１、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄ、ＢＲＣＡ１及びＢＲＣＡ２遺伝子は、ヒトＥＳ細胞又はヒトｉＰＳ細胞と比較して、優位に発現が高いことが確認された。 Furthermore, with respect to marmoset ES cells, results similar to those of DSY127 cells were obtained in other plural marmoset ES cells. As a result, it was confirmed that the RAD51, RAD51C, RAD51D, BRCA1, and BRCA2 genes were significantly higher in expression in the marmoset ES cells than in the human ES cells or human iPS cells.
　前記の遺伝子群がコードするタンパク質は、相同組換えに関わることから、前記遺伝子の高い発現レベルが、マーモセットＥＳ細胞における高い相同組換え効率に寄与している可能性が考えられた。 (4) Since the proteins encoded by the above genes are involved in homologous recombination, it was considered that high expression levels of the genes may contribute to high homologous recombination efficiency in marmoset ES cells.
［実験例３］
（相同組換え効率の制御）
　実験例２に示されたマーモセットで高発現するＤＮＡ切断修復関連タンパク質である、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１ＤをヒトｉＰＳ細胞において発現させて、相同組換え効率への影響を検討した。 [Experimental example 3]
(Control of homologous recombination efficiency)
BRCA1, BRCA2, RAD51C and RAD51D, which are DNA break repair-related proteins highly expressed in marmosets shown in Experimental Example 2, were expressed in human iPS cells, and the effect on homologous recombination efficiency was examined.
《相同組換え効率の定量化》
　まず、相同組換え効率を定量化するために、ヒトｉＰＳ細胞において、ｈｙｐｏｘａｎｔｈｉｎｅ　ｐｈｏｓｐｈｏｒｉｂｏｓｙｌ　ｔｒａｎｓｆｅｒａｓｅ（ＨＰＲＴ）遺伝子座にネオマイシン耐性遺伝子カセットを相同組換えにより挿入する実験系を用いた。この相同組換えの概略を図１に示した。 《Quantification of homologous recombination efficiency》
First, in order to quantify the efficiency of homologous recombination, an experimental system in which a neomycin resistance gene cassette was inserted by homologous recombination into a human iPS cell at the locus of hypoxanthine phosphoribosyl transferase (HPRT) was used. The outline of this homologous recombination is shown in FIG.
　図１中、ＨＰＲＴＷＴは、正常なヒトＨＰＲＴ遺伝子座を示し、ＨＰＲＴ　ｔａｒｇｅｔｉｎｇ　ｖｅｃｔｏｒは、ネオマイシン耐性遺伝子を含むターゲティングベクターを示し、ＨＰＲＴＮｅｏは、相同組換え後のＨＰＲＴ遺伝子座を示す。 In FIG. 1, HPRT WT indicates a normal human HPRT locus, HPRT targeting vector indicates a targeting vector containing a neomycin resistance gene, and HPRT Neo indicates an HPRT locus after homologous recombination.
　ＨＰＲＴが正常である細胞をグアニン類似体の６－チオグアニン（６－ＴＧ）存在下で培養すると、ＤＮＡに６－ＴＧが誤って取り込まれてしまうため細胞は死滅するが、ＨＰＲＴ遺伝子が破壊された細胞は６－ＴＧ存在下でも生存可能である。 When cells with normal HPRT were cultured in the presence of the guanine analog, 6-thioguanine (6-TG), the cells were killed because 6-TG was erroneously incorporated into DNA, but the HPRT gene was destroyed. Cells can survive in the presence of 6-TG.
　また、ゲンタマイシン類似体のＧ４１８は、真核生物のタンパク質合成を阻害して細胞を死滅させるが、ネオマイシン耐性遺伝子が挿入され形質転換した細胞は、Ｇ４１８存在下でも生存可能である。 ゲ ン Although the gentamicin analog G418 inhibits eukaryotic protein synthesis and kills cells, cells transformed with the neomycin resistance gene inserted can survive in the presence of G418.
　また、ゲノムＤＮＡを切断する酵素として、Ｃａｓ９タンパク質を用いた。ガイドＲＮＡの配列はＨＰＲＴ遺伝子のエクソン２を標的とするものを用いた。ガイドＲＮＡの標的配列の塩基配列を配列番号１に示す。Ｃａｓ９タンパク質とガイドＲＮＡを、それぞれ、ＣＡＧプロモーター又はＵ６プロモーター制御下で発現させるＤＮＡコンストラクトを作製した。 Ｃ Further, Cas9 protein was used as an enzyme for cleaving genomic DNA. As the sequence of the guide RNA, one targeting exon 2 of the HPRT gene was used. The nucleotide sequence of the target sequence of the guide RNA is shown in SEQ ID NO: 1. A DNA construct for expressing the Cas9 protein and the guide RNA under the control of the CAG promoter or U6 promoter, respectively, was prepared.
　また、ＨＰＲＴ遺伝子座に相同な配列とネオマイシン耐性遺伝子カセットを有するドナーＤＮＡを作製した。ドナーＤＮＡの塩基配列を配列番号２に示す。ドナーＤＮＡは環状二本鎖ＤＮＡを用いた。 ド ナ ー In addition, a donor DNA having a sequence homologous to the HPRT locus and a neomycin resistance gene cassette was prepared. The nucleotide sequence of the donor DNA is shown in SEQ ID NO: 2. As the donor DNA, a circular double-stranded DNA was used.
　前記のＤＮＡ切断修復関連タンパク質の発現コンストラクト、ドナーＤＮＡ、Ｃａｓ９タンパク質とガイドＲＮＡの発現コンストラクトを混合し、エレクトロポレーション法を用いて野生型ヒトｉＰＳ細胞へ導入した。 (4) The expression construct of the above-mentioned DNA cleavage repair-related protein, the donor DNA, the Cas9 protein and the expression construct of the guide RNA were mixed, and introduced into wild-type human iPS cells by electroporation.
　前記ＤＮＡを細胞へ導入した後、これら細胞を等量の２群に分け、一方にはＧ４１８のみを加え、他方にはＧ４１８と６－ＴＧを加えて、培養し、選別を行った。この２群のコロニー数をそれぞれ計測した。 After the DNA was introduced into the cells, the cells were divided into two equal groups, one of which was supplemented with G418 alone and the other of which was supplemented with G418 and 6-TG, cultured and sorted. The number of colonies in each of the two groups was counted.
　ここで、ネオマイシン耐性遺伝子カセットがゲノム上のいずれかの位置に挿入されたＧ４１８耐性クローンの数は、相同組換え体と非相同組換え体の合計の数を反映する。一方、ネオマイシン遺伝子カセットがＨＰＲＴ遺伝子座に挿入された、Ｇ４１８と６－ＴＧの両方に耐性を持つクローンの数は、相同組換え体の数を反映する。 Here, the number of G418-resistant clones in which the neomycin resistance gene cassette was inserted at any position on the genome reflects the total number of homologous recombinants and non-homologous recombinants. On the other hand, the number of clones resistant to both G418 and 6-TG in which the neomycin gene cassette was inserted at the HPRT locus reflects the number of homologous recombinants.
　そして、Ｇ４１８と６－ＴＧの両方に耐性を持つ細胞と、Ｇ４１８耐性細胞のコロニー数の比を下記式（３）から算出することにより、相同組換え効率を算出することができる。
（相同組換え効率）＝（Ｇ４１８と６－ＴＧの両方に耐性を持つ細胞のコロニー数）／（Ｇ４１８耐性細胞のコロニー数）　（３） Then, the homologous recombination efficiency can be calculated by calculating the ratio of the number of colonies of cells resistant to both G418 and 6-TG to the number of colonies of G418-resistant cells from the following equation (3).
(Homologous recombination efficiency) = (number of colonies of cells resistant to both G418 and 6-TG) / (number of colonies of G418-resistant cells) (3)
《ＤＮＡ切断修復関連タンパク質の過剰発現》
　ヒトＲＡＤ５１、ヒトＲＡＤ５１Ｃ及びヒトＲＡＤ５１Ｄをコードする遺伝子配列を、それぞれ、ＣＡＧプロモーターの下流に連結した発現コンストラクトを作製した。また、ヒトＢＲＣＡ１及びヒトＢＲＣＡ２をコードする遺伝子配列を、それぞれ、ＣＭＶプロモーターの下流に連結した発現コンストラクトを作製した。 << Overexpression of DNA break repair-related proteins >>
Expression constructs were prepared in which gene sequences encoding human RAD51, human RAD51C and human RAD51D were respectively linked downstream of the CAG promoter. In addition, an expression construct in which gene sequences encoding human BRCA1 and human BRCA2 were respectively linked to the downstream of the CMV promoter was prepared.
　ＲＡＤ５１のｍＲＮＡのＮＣＢＩアクセッション番号はＮＭ＿００２８７５．４であり、ＲＡＤタンパク質のＮＣＢＩアクセッション番号はＮＰ＿００２８６６．２である。ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１ＤのＮＣＢＩアクセッション番号は表１と２に示した通りである。 The NCBI accession number of the RAD51 mRNA is NM — 0028755.4, and the NCBI accession number of the RAD protein is NP — 0028666.2. The NCBI accession numbers of BRCA1, BRCA2, RAD51C and RAD51D are as shown in Tables 1 and 2.
　複数のＤＮＡ切断修復関連タンパク質を発現させる場合には、複数の前記ＤＮＡ切断修復関連タンパク質の発現コンストラクトを、前記ドナーＤＮＡコンストラクト、ゲノムＤＮＡを切断する酵素の発現コンストラクトと共に混合し、ヒトｉＰＳ細胞に導入した。 When expressing a plurality of DNA-cutting repair-related proteins, the expression constructs of the plurality of DNA-cutting repair-related proteins are mixed with the donor DNA construct and the expression construct of an enzyme that cuts genomic DNA, and introduced into human iPS cells. did.
　前記ＤＮＡを細胞へ導入した後、これら細胞を等量の２群に分け、一方にはＧ４１８のみを加え、他方にはＧ４１８と６－ＴＧを加えて、培養し、選別を行った。この２群のコロニー数をそれぞれ計測した。 After the DNA was introduced into the cells, the cells were divided into two equal groups, one of which was supplemented with G418 alone and the other of which was supplemented with G418 and 6-TG, cultured and sorted. The number of colonies in each of the two groups was counted.
　計測したコロニー数から、上記式（３）を用いて、相同組換え効率を算出した。結果を図７と図８に示す。図７と図８の縦軸は、上記式（３）により算出した相同組換え効率を示す。図７は１種類の、図８は２種以上のＤＮＡ切断修復関連タンパク質を発現させた場合の相同組換え効率を示す。図７及び図８中、ｍｏｃｋはＤＮＡ切断修復関連タンパク質を発現させなかった場合を示す。 相同 Homologous recombination efficiency was calculated from the measured number of colonies using the above formula (3). The results are shown in FIG. 7 and FIG. The vertical axis in FIGS. 7 and 8 indicates the homologous recombination efficiency calculated by the above equation (3). FIG. 7 shows the homologous recombination efficiency when one or two or more DNA break repair-related proteins are expressed. In FIGS. 7 and 8, mock shows the case where the DNA break repair-related protein was not expressed.
　図７に示す結果から、ＣＭＶプロモーター制御下にヒトＢＲＣＡ１及びヒトＢＲＣＡ２を発現するヒトｉＰＳ細胞は、野生型ヒトｉＰＳ細胞に比べて、相同組換え効率が上昇することが明らかになった。 (7) The results shown in FIG. 7 revealed that the homologous recombination efficiency of human iPS cells expressing human BRCA1 and human BRCA2 under the control of the CMV promoter was higher than that of wild-type human iPS cells.
　図８に示す結果から、ＤＮＡ切断修復関連タンパク質を組み合わせて発現させた場合、すなわちＢＲＣＡ１とＢＲＣＡ２、ＲＡＤ５１ＣとＲＡＤ５１Ｄ、ＢＲＣＡ１とＢＲＣＡ２とＲＡＤ５１ＣとＲＡＤ５１Ｄを発現させた場合、相同組換え効率は有意に上昇することが明らかになった。 From the results shown in FIG. 8, the homologous recombination efficiency is significantly increased when the DNA break repair-related proteins are expressed in combination, that is, when BRCA1 and BRCA2, RAD51C and RAD51D, and BRCA1 and BRCA2, RAD51C and RAD51D are expressed. It turned out to be.
　これらの結果から、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄは、相同組換え効率上昇に寄与することが明らかになった。 These results revealed that BRCA1, BRCA2, RAD51C and RAD51D contribute to an increase in homologous recombination efficiency.
［実験例４］
　マウスＥＳ細胞に、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄ、ＲＡＤ５１を発現させ、Ｓｏｘ２遺伝子座にＶｅｎｕｓ遺伝子を相同組換えにより挿入して、相同組換え効率を算出した。 [Experimental example 4]
BRCA1, BRCA2, RAD51C, RAD51D, and RAD51 were expressed in mouse ES cells, and the Venus gene was inserted into the Sox2 locus by homologous recombination to calculate the homologous recombination efficiency.
　相同組換え効率を定量化するために、マウスＥＳ細胞において、Ｓｏｘ２遺伝子座に緑色蛍光タンパク質であるＶｅｎｕｓを相同組換えにより挿入する実験系を利用した。この相同組換えの実験の概略を図９に示した。 (4) To quantify the homologous recombination efficiency, an experimental system in which Venus, a green fluorescent protein, was inserted into the Sox2 locus by homologous recombination in mouse ES cells was used. The outline of the experiment of this homologous recombination is shown in FIG.
　図９中、Ｗｉｌｄ　ｔｙｐｅはゲノムＤＮＡ上のＳｏｘ２遺伝子座を示し、Ｋｎｏｃｋ－ｉｎは、ＶｅｎｕｓがＳｏｘ２遺伝子座にインフレームで挿入された、相同組換え後のＳｏｘ２遺伝子座を示す。 In FIG. 9, Wild type indicates the Sox2 locus on the genomic DNA, and Knock-in indicates the Sox2 locus after homologous recombination in which Venus was inserted in-frame at the Sox2 locus.
　ゲノムＤＮＡを切断する酵素としてＣａｓ９タンパク質を用いた。また、ガイドＲＮＡは、ＳＯＸ２遺伝子の終止コドン周辺を標的とするように設計した。また、マウスＥＳ細胞として、ＥＢ３株を用いた。 Ｃ The Cas9 protein was used as an enzyme that cuts genomic DNA. The guide RNA was designed to target around the stop codon of the SOX2 gene. The EB3 strain was used as mouse ES cells.
　ドナーＤＮＡとして、ＶｅｎｕｓをコードするＯＲＦと、ＳＯＸ２遺伝子の終止コドンの上流の約５００塩基対と、ＳＯＸ２遺伝子の終止コドンの下流の約５００塩基対とを有するものを作製した。ＶｅｎｕｓのＯＲＦは、ＳＯＸ２遺伝子とインフレームで連結するように、ドナーＤＮＡを設計した。ドナーＤＮＡは環状二本鎖ＤＮＡを用いた。 A donor DNA having an ORF encoding Venus, about 500 base pairs upstream of the stop codon of the SOX2 gene, and about 500 base pairs downstream of the stop codon of the SOX2 gene was prepared. The Venus ORF designed the donor DNA to ligate in frame with the SOX2 gene. As the donor DNA, a circular double-stranded DNA was used.
　ＤＮＡ切断修復関連タンパク質の発現コンストラクトとして、ヒトＢＲＣＡ１遺伝子、ヒトＢＲＣＡ２遺伝子、ヒトＲＡＤ５１Ｃ遺伝子、ヒトＲＡＤ５１Ｄ遺伝子、ヒトＲＡＤ５１遺伝子を、それぞれ、ＣＡＧプロモーターの下流に連結した発現コンストラクトを用いた。 As an expression construct for a DNA cleavage repair-related protein, an expression construct in which a human BRCA1 gene, a human BRCA2 gene, a human RAD51C gene, a human RAD51D gene, and a human RAD51 gene were respectively linked to the downstream of a CAG promoter was used.
　ＤＮＡ切断修復関連タンパク質の発現コンストラクトと、ドナーＤＮＡと、Ｃａｓ９タンパク質及びガイドＲＮＡの発現コンストラクトとを混合したものを、エレクトロポレーション法によりマウスＥＳ細胞へ導入した。続いて、得られた細胞を播種して培養した。結果を図１０に示す。 A mixture of an expression construct for a DNA cleavage repair-related protein, a donor DNA, and an expression construct for Cas9 protein and guide RNA was introduced into mouse ES cells by electroporation. Subsequently, the obtained cells were seeded and cultured. The results are shown in FIG.
　図１０は、相同組換え後、Ｖｅｎｕｓを発現する細胞を撮影した顕微鏡像である。その結果、相同組換えによりＶｅｎｕｓがＳｏｘ２遺伝子座に挿入された細胞においては、ＳＯＸ２：：Ｖｅｎｕｓ融合タンパク質が核に局在することが明らかになった。 FIG. 10 is a micrograph of cells expressing Venus after homologous recombination. As a result, it was revealed that in cells in which Venus was inserted into the Sox2 locus by homologous recombination, the SOX2 :: Venus fusion protein was localized in the nucleus.
　また、エレクトロポレーションを行った細胞を培養し、コロニーを形成させた。形成されたコロニーのうち、Ｖｅｎｕｓを発現する細胞を有するコロニーの割合（Ｖｅｎｕｓ陽性コロニー率）を計測した。結果を図１１に示す。 The cells subjected to electroporation were cultured to form colonies. Among the formed colonies, the ratio of colonies having Venus-expressing cells (Venus positive colony ratio) was measured. The results are shown in FIG.
　図１１は、ＤＮＡ切断修復関連タンパク質を発現させた場合の、Ｖｅｎｕｓ陽性コロニー率を示す結果である。図１１中、４Ｆは、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄを同時に発現させた場合を示し、５Ｆは、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄ及びＲＡＤ５１を同時に発現させた場合を示す。図１１中、ＭｏｃｋはＤＮＡ切断修復関連タンパク質を発現させなかった場合を示す。 FIG. 11 shows the results showing the ratio of Venus-positive colonies in the case of expressing the DNA cleavage repair-related protein. In FIG. 11, 4F shows the case where BRCA1, BRCA2, RAD51C and RAD51D are simultaneously expressed, and 5F shows the case where BRCA1, BRCA2, RAD51C, RAD51D and RAD51 are simultaneously expressed. In FIG. 11, Mock shows the case where the DNA break repair-related protein was not expressed.
　その結果、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄを単独で発現させた場合、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ及びＲＡＤ５１Ｄを発現させた場合、並びに、ＢＲＣＡ１、ＢＲＣＡ２、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄ及びＲＡＤ５１を発現させた場合、ＤＮＡ切断修復関連タンパク質を発現させなかった場合と比較して、Ｖｅｎｕｓ陽性コロニー率が有意に上昇した。 As a result, when BRCA1, BRCA2, RAD51C, RAD51D were expressed alone, when BRCA1, BRCA2, RAD51C, and RAD51D were expressed, and when BRCA1, BRCA2, RAD51C, RAD51D, and RAD51D were expressed, DNA cleavage was observed. The Venus-positive colony rate was significantly increased compared to the case where the repair-related protein was not expressed.
　本発明によれば、相同組換え効率を改善する技術を提供することができる。 According to the present invention, a technique for improving the efficiency of homologous recombination can be provided.

Claims (10)

  1. 　マーモセットの細胞と比較して相同組換え効率が低い種の細胞よりも、マーモセットの細胞における発現量が高い、ＤＮＡ切断修復関連タンパク質又は前記タンパク質をコードする核酸を有効成分として含有する、相同組換え効率上昇剤。 Homologous recombination containing a DNA break repair-related protein or a nucleic acid encoding the protein as an active ingredient, which has a higher expression level in a marmoset cell than a cell of a species having a lower homologous recombination efficiency as compared to a marmoset cell. Efficiency enhancer.
  2. 　マーモセットの細胞と比較して相同組換え効率が低い前記種が、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択される、請求項１に記載の相同組換え効率上昇剤。 Said species having a lower homologous recombination efficiency as compared to marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. The agent for increasing the efficiency of homologous recombination according to claim 1.
  3. 　前記ＤＮＡ切断修復関連タンパク質が、ＲＡＤ５１パラログ及び乳癌易罹患性に関わる相同組換え因子である請求項１または２に記載の相同組換え効率上昇剤。 (3) The agent for increasing the efficiency of homologous recombination according to claim 1 or 2, wherein the DNA breakage repair-related protein is a RAD51 paralog and a homologous recombination factor involved in susceptibility to breast cancer.
  4. 　前記ＤＮＡ切断修復関連タンパク質が、ＲＡＤ５１Ｃ、ＲＡＤ５１Ｄ、ＢＲＣＡ１、ＢＲＣＡ２からなる群より選択される、請求項３に記載の相同組換え効率上昇剤。 剤 The homologous recombination efficiency increasing agent according to claim 3, wherein the DNA cleavage repair-related protein is selected from the group consisting of RAD51C, RAD51D, BRCA1, and BRCA2.
  5. 　相同組換え効率が上昇した細胞の製造方法であって、
    　前記細胞はマーモセットの細胞と比較して相同組換え効率が低い種の細胞であり、
    　前記細胞に請求項１～４のいずれか一項に記載の相同組換え効率上昇剤を導入することを含む、製造方法。     A method for producing a cell having an increased homologous recombination efficiency,
    The cells are cells of a species having a lower homologous recombination efficiency as compared to marmoset cells,
    A production method, comprising introducing the homologous recombination efficiency increasing agent according to any one of claims 1 to 4 into the cells.
  6. 　マーモセットの細胞と比較して相同組換え効率が低い前記種が、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択される、請求項５に記載の製造方法。 Said species having a lower homologous recombination efficiency as compared to marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. The method according to claim 5.
  7. 　請求項１～４のいずれか一項に記載の相同組換え効率上昇剤が導入された、マーモセットの細胞と比較して相同組換え効率が低い種の細胞。 {Circle around (5)} A cell of a species into which the homologous recombination efficiency increasing agent according to any one of claims 1 to 4 has been introduced, which has a lower homologous recombination efficiency than marmosets cells.
  8. 　マーモセットの細胞と比較して相同組換え効率が低い前記種が、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択される、請求項７に記載の細胞。 Said species having a lower homologous recombination efficiency as compared to marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. The cell according to claim 7.
  9. 　相同組換えによりゲノムＤＮＡが改変された細胞の製造方法であって、
    　前記細胞はマーモセットの細胞と比較して相同組換え効率が低い種の細胞であり、
    　前記細胞に請求項１～４のいずれか一項に記載の相同組換え効率上昇剤を導入することと、
    　前記細胞にドナーＤＮＡを導入することと、
    　前記細胞のゲノムＤＮＡを切断することと、
    　を含む、製造方法。     A method for producing a cell in which genomic DNA has been modified by homologous recombination,
    The cells are cells of a species having a lower homologous recombination efficiency as compared to marmoset cells,
    Introducing the homologous recombination efficiency increasing agent according to any one of claims 1 to 4 into the cells;
    Introducing a donor DNA into the cells;
    Cutting the genomic DNA of the cell;
    And a manufacturing method.
  10. 　マーモセットの細胞と比較して相同組換え効率が低い前記種が、ヒト、マウス、ラット、ウサギ、サル、ブタ、ヤギ、ヒツジ、ウシ、フェレット、イヌ、ネコ及びニワトリからなる群より選択される、請求項９に記載の製造方法。 Said species having a lower homologous recombination efficiency as compared to marmoset cells is selected from the group consisting of human, mouse, rat, rabbit, monkey, pig, goat, sheep, cow, ferret, dog, cat and chicken. The method according to claim 9.
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